DFT Study on the His-Tag Binding Affinity of Metal Ions in Modeled Hexacationic Metal Complexes

Histidine oligomers (His-tags) are commonly used as affinity tags in recombinant protein purification to enable in vitro experimental studies, including biochemical and biophysical assays and structure determination. His-tags enable protein purification by specifically and efficiently coordinating bivalent metal ions present in the purification resins, such as Cu²⁺, Zn²⁺, and Ni²⁺. Although His-tags, combined with Ni²⁺-based resins, are widely used due to their biophysical properties and commercial availability, the structure and nature of the metal cation coordination have remained unclear. In this study, the chemical structure of metal-coordinating His-tags was modeled to elucidate the metal preferences for better binding and to determine the structural changes that occur upon metal coordination. 6His-tag is a string of 6 histidine residues usually coordinated to bivalent metal ions (M²⁺), such as Ni²⁺, Zn²⁺, and Cu²⁺, through the nitrogen atoms of the imidazole rings. The metals complete their octahedral coordination shell with the lone pair of electrons of the oxygen atoms of the resin to which they are attached to. The resin was modeled as an oxalaldehyde group with the closed formula of (OCH)₄. The geometry optimizations were carried out using the DFT method at the B3LYP/6-31g (d, p) level and in implicit water using the IEFPCM solvent model. The impact of Ni²⁺, Zn²⁺, and Cu²⁺ metals on the resin binding ability of 6His-tags was tested by adding amino acids to the N-terminus of metal-coordinated hexahistidine chains. The activity of the metals as electron donors or acceptors to the coordination bonds that they form was estimated by calculating the NBO energies. The complexation enthalpies of metal-bearing resin with hexahistidine, naked or having an amino acid tail, were calculated. Our results showed that Ni²⁺ has the highest affinity for the recombinant 6His-tag.